- Email: [email protected]
Mycobacterium marinum Infection in Japanese Forest Green Tree Frogs (Rhacophorus arboreus)
J. Comp. Path. 2014, Vol. 151, 277e289
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
DISEASE IN WILDLIFE OR EXOTIC SPECIES
Mycobacterium marinum Infection in Japanese Forest Green Tree Frogs (Rhacophorus arboreus) M. Haridy*,†, Y. Tachikawa‡, S. Yoshidax,k, K. Tsuyuguchix, M. Tomitax, S. Maeda{, T. Wadak, K. Ibi*, H. Sakai* and T. Yanai* * Department of Pathogenetic Veterinary Sciences, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan, † Department of Pathology and Clinical Pathology, Faculty of Veterinary Medicine, South Valley University, Qena 83523, Egypt, ‡ Gifu World Fresh Water Aquarium, Gifu, x National Hospital Organization, Kinki-Chuo Chest Medical Centre, Sakai, Osaka 591-8555, k Institute of Tropical Medicine, Nagasaki University, Nagasaki and { The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama, Kiyose-shi, Tokyo 204-8533, Japan
Summary Four Japanese forest green tree frogs (Rhacophorus arboreus) were presented with emaciation, abdominal distention and ulcerative and nodular cutaneous lesions affecting the brisket, limbs, digits and ventral abdomen. Another three frogs had been found dead in the same tank 1 year previously. Necropsy examination of these seven frogs revealed splenomegaly and hepatomegaly, with multiple taneyellow nodular foci present in the liver, spleen, heart, lungs, ovaries and kidneys. Microscopically, five frogs had necrosis and surrounding granulomatous inflammation in the liver, spleen, kidneys, lungs, intestine and ovaries, with numerous acid-fast bacilli in the areas of necrosis. Two frogs had granulomatous lesions in the lungs, liver, spleen, heart, coelomic membrane, stomach and intestinal wall. These lesions had no or minimal necrosis and few acid-fast bacilli. Mycobacterium spp. was cultured from three frogs and identified as Mycobacterium marinum by colony growth rate and photochromogenicity and DNA sequencing. This is the first report of M. marinum infection in Japanese forest green tree frogs. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: Japanese forest green tree frog; mycobacteriosis; Mycobacterium marinum; Rhacophorus arboreus
Introduction Japanese forest green tree frogs (Rhacophorus arboreus), designated as a ‘special natural monument’ in Japan, inhabit Honshu Island in forests, near rice fields and in marshy land. They live in mountainous forests as high as 2,000 m. During the breeding season, the frogs can be found near ponds and lakes, but they live primarily in trees in forests and are the only frogs in Japan that lay their eggs in trees. They are nocturnal and native to Japan. Their predators include snakes, weasels, foxes, raccoon dogs and badgers (Wilkinson, 2003). Most studies of Japanese forest green tree frogs have focused on their reproductive biology and ecol-
ogy (Kusano et al., 2006; Muto and Kubota, 2011). Japanese forest green tree frogs have been reported in the International Union for the Conservation of Nature (IUCN) Red List of threatened species as being of ‘least concern’ (Kaneko and Matsui, 2004). Mycobacterium marinum is a slow-growing environmental mycobacterium that is distributed widely in aquatic environments, especially in fish tanks and swimming pools, and is the most common species associated with tuberculous granulomas in aquariumhoused and wild fish (Gauthier and Rhodes, 2009). M. marinum is a zoonotic agent, which may cause granulomatous lesions on the hands and feet of people following exposure of abraded skin to infected water. Deeper spread of infection may lead to tenosynovitis, arthritis, bursitis and osteomyelitis. Systemic infection
*Correspondence to: T. Yanai (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.04.014
Ó 2014 Elsevier Ltd. All rights reserved.
278
M. Haridy et al.
is rare and always occurs in immunosuppressed individuals (Lewis et al., 2003). M. marinum infection is described infrequently in frogs, although frogs, snakes and turtles have been considered a source of infection for fish (Decostere et al., 2004). M. marinum causes chronic granulomatous non-lethal disease in immunocompetent leopard frogs (Rana pipiens) and an acute, fulminant lethal disease in immunosuppressed frogs (Ramakrishnan et al., 1997). M. marinum was also reported as the causative agent of an outbreak of mycobacteriosis in bullfrogs (Rana catesbeiana) in a commercial breeding farm in Brazil. The infection in that outbreak was characterized by skin lesions and disseminated granulomatous lesions in both symptomatic and asymptomatic frogs (Ferreira et al., 2006). Other non-tuberculous mycobacteria, including Mycobacterium chelonae, Mycobacterium xenopi and Mycobacterium liflandii, have been isolated from diseased South African clawed frogs (Xenopus laevis) (Green et al., 2000; Godfrey et al., 2007) and Mycobacterium gordonae, Mycobacterium szulgai and M. liflandii have been isolated from diseased western clawed frogs (Xenopus tropicalis) (Chai et al., 2006; Suykerbuyk et al., 2007; SanchezMorgado et al., 2009; Fremont-Rahl et al., 2011). South African and western clawed frogs are popular laboratory amphibian models. Mycobacterium ulcerans is related genetically to M. marinum and genomic sequencing has confirmed the evolution of M. ulcerans from a common M. marinum progenitor (Doig et al., 2012). The resolution power of 16S rRNA sequences alone is often insufficient for comparing closely-related organisms (Palys et al., 1997). Protein-encoding genes may be more discriminatory than rRNA-encoding genes (Palys et al., 1997). The genes encoding the 65 kD heat shock protein (hsp65) and b subunit of bacterial RNA polymerase (rpoB) are present in all Mycobacterium spp., but there are species-specific variations (Devulder et al., 2005). The production of frog meat by raniculture has become an important economic activity in many countries, yet little is known about frog diseases and the role of frogs in the maintenance and dissemination of zoonotic diseases in production farms and exhibition aquaria. M. marinum infection in frogs and fish is used as a model for the study of human tuberculosis in order to understand the basic aspects of Mycobacteriumehost interaction and granuloma development, as well as trafficking of immune cells in host tissues (Cosma et al., 2006). In the present study, we describe the pathological findings of M. marinum infection in a new host, the Japanese forest green tree frog (R. arboreus), in an exhibition aquarium in Japan.
Materials and Methods Case Material and Clinical History
In September 2013, a dead Japanese forest green tree frog (case 1) was sent from an exhibition aquarium in Gifu Prefecture to the Laboratory of Veterinary Pathology, Gifu University. The frog was emaciated, skinless and had dark, pinpoint, brown discolouration of the musculature. One week later, another three frogs from the same aquarium were presented. One moribund frog had a distended abdomen and died 15 h after arrival (case 2). The other two frogs were in good body condition, but one had ulcerative and nodular cutaneous lesions affecting the brisket, limbs, digits and ventral abdomen (case 3) and the other had no apparent lesions (case 4). Three further frogs had been found dead in the same tank at the exhibition aquarium 1 year previously. These had been preserved in 10% neutral buffered formalin and were submitted at the same time as cases 1e4. One of these fixed frogs had white discolouration of one eye (case 5), but the other two frogs had no external lesions (cases 6 and 7). Histopathology
Tissue samples of the internal organs were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections were stained by haematoxylin and eosin (HE) and by the ZiehleNeelsen (ZN) stain for acid-fast bacteria when mycobacteriosis was suspected. Cytological impression smears of liver, spleen, kidney and coelomic effusion were stained by WrighteGiemsa stain. Bacteriology
Tissue specimens from three frogs (cases 2e4), including liver, kidney and spleen, were removed aseptically for bacteriological examination. Smears of these tissue samples were stained by ZN and the fluorescent auramineerhodamine staining method and the stained specimens were scored according to the recommendations of the American Thoracic Society (2000). The tissue specimens were homogenized in water and treated with N-acetyl-L-cysteinesodium hydroxide for decontamination. They were then inoculated into 1% Ogawa medium (Kyokuto Pharmaceuticals Industrials Co. Ltd, Tokyo, Japan) and a Mycobacterium growth-indicator tube (MGIT) (Becton Dickinson and Co., Franklin Lakes, New Jersey, USA) and incubated at 25 C and 36 C for 8 weeks. The photochromogenicity of the
Mycobacteriosis in Green Tree Frogs
mycobacterial colonies growing on Ogawa medium was checked daily after exposure to light. DNA Sequencing
Several subcultured colonies were suspended in 100 ml of sterile distilled water and heat-denatured in boiling water for 10 min. The suspensions were used as polymerase chain reaction (PCR) templates for sequencing analysis. Four DNA sequences, the entire internal transcribed spacer (ITS), partial 16S rDNA (477 of N terminus), hsp65 (401 base pairs [bp]) and rpoB (397 bp), were determined using methods described elsewhere (Telenti et al., 1993; Devulder et al., 2005). The sequences obtained were compared with sequences from the DNA Data Bank of Japan (DDBJ) using BLAST (http://blast.ddbj.nig.ac.jp/ blastn). The hsp65 and rpoB sequences were also analyzed using CLUSTALW (Thompson et al., 1994) for multiple alignments with those genes from reference strains of M. marinum and M. ulcerans Agy99.
Results Gross Pathology
Case 1 was emaciated with pinpoint dark haemorrhagic foci in the muscles of the back and hindlimbs. Additionally, there was hepatomegaly, mottled discolouration of the liver and kidney and pinpoint white foci in the spleen. Case 2 had a distended abdomen with coelomic effusion, hepatosplenomegaly, a mottled discoloured liver (Fig. 1a) and multiple taneyellow foci in the spleen, heart and ovary (Fig. 1b). Skin lesions in case 3 were multifocal, primarily involving the limbs, dorsum and ventral abdomen. The lesions were discrete, raised, tanered, nodular foci (0.5e1.5 mm) on the dorsum and ventral abdomen (Fig. 1c), as well as smooth tanered glistening abrasions and ulcers on the digits, limbs, brisket and ventral abdomen (Fig. 1d). Multifocal to coalescing tanewhite foci were observed in the peritoneum, liver, spleen and testis (Fig. 1e). Case 4 was apparently healthy; however, necropsy examination revealed the presence of several whiteetan foci in the liver and lungs (Fig. 1f). A few whiteetan pinpoint foci were also observed in the kidney, heart and stomach in case 5. Marked hepatomegaly and prominent yellow foci in the lungs and heart were observed in case 6, and a mottled discoloured liver in case 7. Splenomegaly and hepatomegaly were present in five cases. On cross sectioning, the splenic and hepatic parenchyma had distinct, variably sized, multifocal to coalescing taneyellow and white foci (0.5e2.0 mm).
279
Cytology and Histopathology
Smears of coelomic fluid from two frogs (cases 2 and 5) were moderately cellular. Nucleated cells were predominantly large mononuclear cells interpreted as being macrophages. The macrophages had abundant vacuolated cytoplasm. Numerous rod-shaped to filamentous, negatively-stained, slender, linear bacilli, approximately 5.5 1.25 mm and consistent with Mycobacterium spp., were observed in the cytoplasm of macrophages and extracellularly (Fig. 2a). Multiple foci of necrosis and granulomatous inflammation were observed in the liver, spleen, kidneys, lungs, intestine and ovaries in five cases (1, 2 and 5e7), with abundant acid-fast bacilli in the centre of the lesions (Fig. 2b). These lesions were characterized by a central necrotic core surrounded by few macrophages (Fig. 2c). The periphery of the necrotic core was stained deeply by ZN stain. Dispersed acidfast bacilli were observed in macrophages in the splenic red pulp. The granulomata sometimes appeared as collections of macrophages engulfing numerous bacilli or, alternatively, as small necrotic granulomata constituted mainly of macrophages with a very small necrotic centre containing abundant bacilli (Fig. 2d). The kidney lesions were confined to the interstitium and comprised of either necrotic tissue with minimal macrophage infiltration or a collection of infiltrating cells without necrosis (Fig. 2e). Free bacilli and bacilli within macrophages were observed in the blood vessel lumina. Renal tubules and glomeruli were almost completely free of bacilli. In hepatic tissue, although the predominant lesions were the multiple foci of necrosis and granulomatous inflammation (Fig. 2f), there was also evidence of periductal fibrosis, fatty degeneration and depletion of melanomacrophage centres. A high density of acid-fast bacilli was observed at the junction of the necrotic core with the surrounding cellular zone of macrophages and epithelioid cells (Fig. 2g). Vasculitis, characterized by infiltration of acid-fast bacilli and mononuclear cells into the subintima and tunica media, was also observed (Fig. 2h). Necrosis of blood vessel walls appeared to be a consequence of extension from adjacent tissue inflammatory foci. Emboli of necrotic debris and macrophages containing bacilli were also detected in some blood vessels. Occasional endothelial cells contained acid-fast bacilli. In the lungs, the lesions ranged from focal thickening of the pulmonary interstitium by disorganized collections of macrophages containing acid-fast bacilli to areas of necrosis and granulomatous inflammation replacing the entire interstitium. In cardiac muscles,
M. Haridy et al.
280
a
b
c
d
e
f
Fig. 1. Gross lesions in Japanese forest green tree frogs infected with M. marinum. (a) Hepatomegaly with mottling (case 2). (b) Multiple taneyellow granulomas in the spleen, heart and ovary (case 2). (c and d) Glistening skin ulcers on the dorsum, digits, limbs, brisket and abdomen (case 3). (e) Multiple white foci on the peritoneum, spleen and liver (case 3). (f) Solitary granuloma in the liver and spleen (case 4).
acid-fast bacilli were present within macrophages and were also noted extracellularly in and adjacent to small blood vessels. Tiny granulomas were noted rarely. Haemorrhages between skeletal muscle fibre bundles (case 1) were present, together with macrophages containing acid-fast bacilli in small arteries. In addition to necrotic and granulomatous lesions in the intestinal wall, collections of foamy macrophages were also observed. Acid-fast bacilli were pre-
sent within the necrotic core as well as within the cytoplasm of foamy macrophages. Blood vessels containing macrophage-laden acid-fast bacilli were observed. Degenerate ova with tiny necrotic and granulomatous foci containing macrophages with cytoplasmic acid-fast bacilli were observed in case 2 (Fig. 2i). The lesions observed in the formalin-fixed frogs (cases 5e7) were also characterized by necrosis with
Mycobacteriosis in Green Tree Frogs
a
b
c
d
e
f
g
h
i
j
k
l
281
Fig. 2. Cytological and histopathological appearance of caseous necrotic granulomas in Japanese forest green tree frogs infected with M. marinum. (a) WrighteGiemsa-stained mononuclear cells with abundant vacuolated cytoplasm containing numerous rod-shaped to filamentous, slender and linear bacilli (case 2). Bar, 20 mm. (b) Multiple caseous necrotic granulomata in the spleen showing a central necrotic core (case 2). HE. Bar, 200 mm. (c) Caseous necrotic granuloma in the spleen with minimal surrounding inflammatory cell infiltration (case 2). HE. Bar, 40 mm. (d) Small necrotic granulomas, comprised mainly of macrophages with a small necrotic centre, contain numerous acid-fast bacilli (case 1). ZN. Bar, 40 mm. (e) A lesion of the renal interstitium that is comprised of necrotic tissue with minimal macrophage infiltration (case 1). The area is stained deeply by ZN. Bar, 80 mm. (f) Multiple caseous necrotic granulomata in hepatic tissue (case 1). HE. Bar, 200 mm. (g) A high density of acid-fast bacilli is observed at the periphery of the necrotic core (case 2). ZN. Bar, 40 mm. (h) Vasculitis with infiltration of mononuclear cells lacking acid-fast bacilli in hepatic tissue (case 1). HE. Bar, 40 mm. (i) Degenerate ova with tiny necrotic granulomata in the ovaries (case 2). HE. Bar, 400 mm. (j) A chronic caseous necrotic granuloma encircled by a thick wall of epithelioid cells and fibroblasts (case 5). HE. Bar, 80 mm. (k) Extensive fibroplasia and bile duct proliferation enclosing several melanomacrophage centres associated with multiple extensive areas of necrosis (case 6). HE. Bar, 80 mm. (l) Hypertrophy of the melanomacrophage centres with large foamy macrophages (case 7). HE. Bar, 40 mm.
282
M. Haridy et al.
granulomatous inflammation and extensive fibrosis. In case 5, focal mononuclear cell infiltrations, including macrophages, epithelioid cells and lymphocytes, were observed in the limbus of the eye. Several chronic caseous necrotic granulomas with a thick wall of epithelioid cells and fibrous connective tissue were observed in renal, gastric submucosal and peritoneal tissues (Fig. 2j). Marked destructive caseous necrotic lesions with extensive fibroplasia and bile duct proliferation, enclosing several melanomacrophage centres, were observed in the liver of case 6 (Fig. 2k). Additionally, the lungs and heart of this animal had severe multifocal destructive caseous necrosis. Hypertrophy of the melanomacrophage centres, with large foamy macrophages, was observed in case 7, and sometimes depletion of melanin cells was present (Fig. 2l). Large numbers of acid-fast bacilli were present in the areas of caseous necrosis and in the melanomacrophage centres. Focal lymphocytic infiltration was observed in one or more of the renal, hepatic and cardiac tissues in cases 5, 6 and 7. Mononuclear cell infiltration consisting mainly of lymphocytes was observed in the wall of blood vessels in hepatic tissue. Multifocal epithelioid granulomatous lesions were observed independently in the lungs, liver, kidneys, stomach and intestinal walls, cardiac and skeletal muscles and coelomic membrane in cases 3 and 4. Epithelioid granulomas were characterized by tightly-packed cells with indistinct cytoplasmic borders and abundant eosinophilic cytoplasm with rare or few acid-fast bacilli. There was almost absent or minimal necrosis within these epithelioid granulomas. In the lungs, areas of pulmonary interstitial thickening with epithelioid granulomas appeared as foci of epithelioid cells and occasional foamy macrophages containing few acid-fast bacilli (Fig. 3aec). A few epithelioid granulomas had central necrotic cells with a higher number of acid-fast bacilli. The bacilli were dispersed singly and intracellularly. The pulmonary interstitium was markedly affected and there was focal dystrophic calcification. In the liver, large epithelioid granulomas containing few acid-fast bacilli were associated with enlarged melanomacrophage centres with centrally-located foamy macrophages surrounded by epithelioid cells. These centres contained few or no acid-fast bacilli. There was also extensive epithelioid cell infiltration of the blood vessel wall, some of these cells containing a few acid-fast bacilli. Renal epithelioid granulomas were composed mainly of epithelioid cells and occasional foamy macrophages containing few acid-fast bacilli (Fig. 3d, e). Splenic epithelioid granulomas (Fig. 3f) were composed of mixed proportions of epithelioid cells in the outer layer and foamy macro-
phages containing few acid-fast bacilli in the centre (Fig. 3g). Two large epithelioid granulomas were found in the heart; however, one had a small necrotic centre and relatively more acid-fast bacilli than the other. Granulomatous coelomitis with abundant macrophages and epithelioid cells was observed in case 3 (Fig. 3h). In that animal, the skeletal muscles contained epithelioid granulomas with few acid-fast bacilli (Fig. 3i). In the stomach, a large, solitary granuloma was found in the muscular layer, with a central necrotic core surrounded by foamy macrophages and epithelioid cells containing few acid-fast bacilli. In the tunica muscularis of the intestine, large granulomas composed mainly of macrophages and epithelioid cells with few acid-fast bacilli were observed. Bacteriology and DNA Sequencing
The aetiological agents were cultured from frogs 2, 3 and 4 (Table 1). Several smooth, white colonies (Fig. 4) grew on solid Ogawa medium (incubated at 25 C) within 3 weeks in each case (Table 1). Inoculation into MGIT also led to growth of the causative agents. However, the bacteria in cases 2 and 3 failed to grow at higher temperature (36 C). The colonies turned yellow after exposure to light (photochromogenicity), which is characteristic of M. marinum (Fig. 4). The ITS and 16S rDNA sequences obtained from these organisms were identical to those of a reference strain of M. ulcerans (Agy99) and various strains of M. marinum. The sequences were registered in the GenBank database as AB624295.1 (ITS) and AM884315.1 (16SrDNA) (data not shown). Two housekeeping genes (hsp65 and rpoB) were also analyzed and were shown to be identical in sequence to those of M. marinum, but slightly different to those of M. ulcerans (Fig. 5).
Discussion The present study reports the pathological changes associated with M. marinum infection in Japanese forest green tree frogs. Five frogs had necrotic and granulomatous lesions in the liver, spleen, kidneys, lungs, intestine and ovaries, with numerous acid-fast bacilli in the necrotic centre. In two of these five frogs, a central necrotic core was surrounded by a thin rim of macrophages, while the lesions in the other three animals were more granulomatous in nature and surrounded by a thick wall of epithelioid cells and fibroblastic tissue. Similar epithelioid granulomatous lesions were observed independently in two further frogs in the lungs, liver, spleen, heart, coelomic membrane, stomach and intestinal wall and were
Mycobacteriosis in Green Tree Frogs
a
b
c
d
e
f
g
h
i
283
Fig. 3. Histopathological appearance of epithelioid granulomas in Japanese forest green tree frogs infected with M. marinum. (a) Multifocal epithelioid granulomas in the pulmonary interstitium (case 3). HE. Bar, 400 mm. (b) These are composed mainly of densely-packed epithelioid cells. HE. Bar, 20 mm. (c) They contain few acid-fast bacilli. ZN. Bar, 20 mm. (d) Renal epithelioid granuloma composed primarily of epithelioid cells (case 3). HE. Bar, 40 mm. (e) This contains few acid-fast bacilli. ZN. Bar, 20 mm. (f) Splenic epithelioid granulomata composed of epithelioid and foamy macrophages (case 4). HE. Bar, 200 mm. (g) These contain few acid-fast bacilli. ZN. Bar, 20 mm. (h) Granulomatous coelomitis with abundant macrophages and epithelioid cells (case 3). HE. Bar, 200 mm. (i) Skeletal muscles with an epithelioid granuloma comprised of tightly-packed epithelioid cells (case 3). HE. Bar, 80 mm.
characterized by extensive epithelioid cell formation with no or minimal necrosis and rare or few acidfast bacilli. The discrete well-organized granulomas (‘tuberculoid granulomas’) that occur in human infections with Mycobacterium tuberculosis are usually a result of strong cell-mediated immunity (CMI). Morphologically, tuberculoid granulomas may be simple or complex in organization, usually contain a paucity of bacilli, may or may not have central caseation and are composed predominantly of macrophages, epithelioid macrophages and multinucleated giant cells (Langhans giant cells) that are encircled by lymphocytes, plasma cells and a peripheral rim of fibroblasts
(Williams and Williams, 1983). The epithelioid granulomas caused by M. marinum infection in the present Japanese forest green tree frogs shared characteristics with those of tuberculoid granulomas including a dominant macrophage and epithelioid cell zone containing few acid-fast bacilli, occasionally with peripheral lymphocyte infiltration. No giant cells were observed in any of the frogs examined. Similarly, no giant cells were observed in the granulomas caused by M. marinum and M. liflandii in other species of frogs (Ramakrishnan et al., 1997; Bouley et al., 2001; Ferreira et al., 2006; Fremont-Rahl et al., 2011). The so-called ‘lepromatous granulomas’ arise in a host with robust humoral immunity, but poor CMI
M. Haridy et al.
284 Table 1 Mycobacterial colonies cultured from infected Rhacophorus arboreus 25 C
Case number Specimen Acid-fast stain*
36 C
MGIT Ogawa MGIT Ogawa 2
Spleen Kidney Liver
3+ 3+ 3+
+ + +
+ + +
+ + +
+ +
3
Liver Kidney
2+ 3+
+ +
+ +
4
Liver Kidney
3+ 3+
+ +
+ +
*
ZiehleNeelsen and fluorescent auramineerhodamine staining. For ZN (1,000), a quantitative score of acid-fast bacilli (AFB) was assigned: , no AFB in 300 fields; +, 1e9 AFB in 100 fields; 2+, 1e9 AFB in 10 fields; 3+, 1e9 AFB in one field. For fluorochrome staining (250), quantitative scoring of AFB was used: , no AFB in 30 fields; +, 1e9 AFB in 10 fields; 2+, 1e9 AFB in one field; 3+, 10e90 AFB in one field.
Fig. 4. Morphology (left slant) and photochromogenicity (right slant) of M. marinum colonies on Ogawa medium. White colonies of M. marinum turned yellow when exposed directly to light.
to Mycobacterium spp. These lesions typically lack structural organization and are characterized by diffuse infiltration, mostly of macrophages with numerous intracytoplasmic acid-fast bacilli, sparse lymphocytes and rare to no giant cells (Williams and Williams, 1983). The necrotic granulomata with collections of macrophages containing abundant acid-fast bacilli, and the caseous lesions observed in renal tissue in Japanese forest green tree frogs, shared features of human lepromatous granulomas, including the presence of mature macrophages, epithelioid cells and extracellular components, a fibrous connective tissue capsule and caseation, but the lesions in the frogs lacked giant cells (Bouley et al., 2001). M. marinum can cause caseous necrosis in man, goldfish and the toads Xenopus laevis and Xenopus borealis (Travis et al., 1985; Asfari, 1988; Talaat et al., 1998), but not in leopard frogs (Ramakrishnan et al., 1997; Bouley et al., 2001). In the Japanese forest green tree frogs, the granulomas contained a central caseous necrotic core filled with numerous acid-fast bacilli that was surrounded by a thin rim of epithelioid cells or a thick wall of epithelioid cells and fibroblasts. These granulomas had features similar to those described following experimental inoculation of M. marinum into zebrafish (Swaim et al., 2006), goldfish (Talaat et al., 1998), striped bass (Gauthier et al., 2003) and medaka (Broussard and Ennis, 2007). The epithelioid granulomas were compact and composed of tightly-packed cells that displayed indistinct cytoplasmic borders and abundant eosinophilic cytoplasm, characteristics of epithelioid cells (Bouley et al., 2001). Japanese forest green tree frogs might be a promising animal model for studying tuberculous granulomas because they share both the organized, non-necrotic epithelioid granuloma and the organized necrotic granuloma forms reported in experimental infection with M. marinum of leopard frogs and of zebrafish, respectively. Acid-fast bacilli are observed within the central caseous material and in macrophages of human conventional tuberculous granulomata, and necrotic granulomata containing organisms are seen in active human tuberculosis (Ulrichs and Kaufmann, 2006). Chronic asymptomatic tuberculous infection of man is characterized by fibrotic and calcified granulomas that may or may not contain viable bacteria. Necrosis likely plays an important role in the morbidity and transmission of tuberculosis, but its existence is host and microbial dependant. The necrotic caseous granulomas in Japanese forest green frogs are comparable with the necrotic granulomas of human tuberculosis. A similar high density of acid-fast bacilli is observed in the necrotic centre and epithelioid cell aggregates of
Mycobacteriosis in Green Tree Frogs
285
a
Fig. 5. Multiple alignments of (a) hsp65 and (b) rpoB DNA sequences. Areas of sequence variation are highlighted.
these lesions; however, epithelioid granulomas in frogs also shared some features of human chronic asymptomatic tuberculous infection including low density of acid-fast bacilli, no clinical signs and occasional dystrophic calcification without fibrosis. Measurement of the size of granulomata and calculation of the ratio of the area of the surrounding in-
flammatory cell region to the necrotic core shows that as a granuloma enlarges, the ratio decreases, suggesting that necrosis expands at the expense of the surrounding cell layer rather than being proportional to growth of the granuloma itself (Ulrichs et al., 2004). The necrotic granulomata in the present report had either a wide central necrotic core encircled by a
M. Haridy et al.
286
b
Fig. 5. (continued)
rim of macrophages (cases 1 and 2) or an extensive necrotic area with a thick fibroblastic capsule (case 6), suggesting a late stage of infection. This association was supported by the fatal nature of this type of granuloma in affected frogs. Necrosis within tuberculous granulomas is produced by autodigestion by macrophage enzymes and by a direct toxic action of the causative agent. This process is augmented by
the cellular and humoral immune responses. In the rat model, the main stimulus for necrosis was the formation of immune complexes of antibody and excess bacillus antigen in the centre of lesions. This occurred when CMI was initially strong, but began to decline, allowing the mycobacteria to proliferate out of control of the macrophages. If CMI improved again, the number of bacilli diminished and immune
Mycobacteriosis in Green Tree Frogs
complexes increased, producing an epithelioid granuloma instead of necrosis (Yamamura et al., 1974). The immune status of the frogs in the present report was not examined; however, multifocal areas of lymphocytic infiltration were observed in cases 5, 6 and 7. The acid-fast bacilli in the frogs with necrotic granulomas were found predominantly at the periphery of the necrotic core, adjacent to the epithelioid cell mantle. The same pattern is recognized in human granulomas, where some granulomas harbour mycobacteria within their necrotic cores. Mycobacterium may also be detected in the surrounding region of infiltrating leucocytes, indicating that antigen presentation takes place at the periphery of the classical granuloma structure (Ulrichs et al., 2004). In the present Japanese forest green tree frogs, the presence of epithelioid granulomata characterized by no or minimal necrosis with rare or few acid-fast bacilli suggests a latent infection. A dynamic balance between host immune response and M. tuberculosis is maintained in latent human infections (Ulrichs and Kaufmann, 2002). Dormant M. tuberculosis organisms appear to alter their cell wall structures, since they lose their acid-fast property during latency. In the present study, the tissue sections of the epithelioid and necrotic caseous granulomas of different frogs were stained by ZN at the same time, with the same technique and reagents. This supports a low density of acid-fast bacilli or loss of acid-fast staining of bacteria in epithelioid granulomas. ZN-negative bacilli were detected in chronicallyinfected mice and in non-progressive human tuberculous lung lesions (Zhang, 2004). In the present frogs, lesions in the kidneys were small aggregates of macrophages or tiny necrotic foci confined to the renal interstitium and closely associated with the renal vasculature. Glomerular changes were not detected and both glomeruli and tubules were free of acid-fast bacilli. Small multifocal lymphohistiocytic interstitial aggregates were observed in only one frog out of 23 in a research colony of X. tropicalis infected with M. liflandii (Fremont-Rahl et al., 2011). Tuberculous interstitial nephritis is a common sequela to human renal tuberculosis and glomerular lesions are rarely reported (Ram et al., 2011). In these Japanese forest green tree frogs, acid-fast bacilli were detected in macrophages in the lumina of renal, muscular, cardiac, intestinal and hepatic blood vessels. Extracellular and intracellular acidfast bacilli were observed in the dermal and endomysial vasculature, splenic ellipsoids, renal sinusoids and bronchial vasculature in farmed Atlantic salmon infected with M. chelonae (Brocklebank et al., 2003). Mononuclear cell infiltration of the vascular wall
287
causing vasculitis associated with few or no bacilli was detected in most of the infected frogs. Some endothelial cells of hepatic blood vessels contained acidfast bacilli. Thrombotic lesions associated with numerous mycobacteria have been reported previously in fish with M. marinum infection (Giavenni et al., 1980). Susceptibility to mycobacterial infection depends on the immune status of the host. In immunocompetent leopard frogs inoculated with M. marinum, a chronic granulomatous non-lethal disease was observed. There were at most 1e2 organisms per granuloma. In contrast, leopard frogs administered hydrocortisone intraperitoneally and inoculated with M. marinum exhibited an acute fulminating lethal disease characterized by diffuse chronic inflammatory cell infiltration without granuloma formation or very poorly formed granulomas containing a very high density of M. marinum bacilli (Ramakrishnan et al., 1997). In the present report, necrotic granulomas contained a high density of acid-fast bacilli with little epithelioid recruitment and a typically lethal nature, suggesting severe immunosuppression in the affected frogs. In the other three frogs with an encircling fibrous capsule or fibroplasia, multifocal lymphocytic infiltrations were observed, suggesting that host immunity may be responsible for this encircling of the foci of caseous necrosis with fibroblasts. However, the large number of epithelioid cells in epithelioid granulomas, combined with a scarcity of acid-fast bacilli in these lesions, suggested immunocompetence of the infected frogs. Mycobacterial granulomas in immunosuppressed marsupials may lack such epithelioid and giant cells and fibrous encapsulation (Buddle and Young, 2000). In human tuberculous lymphadenitis, a correlation between the type of granuloma (epithelioid with or without necrosis or necrotic) and the presence and number of acid-fast bacilli revealed that the number of acid-fast bacilli was significantly higher in the presence of necrosis and neutrophilic infiltration when compared with lesions containing lymphocytes, epithelioid cells and Langhans giant cells (Das et al., 1990). Acid-fast bacilli were seen consistently in the intestinal content of frogs 1 and 2, suggesting that food may have been a source of infection. Frogs feed on spiders, insects and sludge worms (Iwai and Kagaya, 2005). M. marinum was demonstrated in the mud tube worm (Tubifex tubifex) that served as live fish food for aquarium fish. The ingestion of these worms by the mangrove killifish was shown to be the source of their infection (Nenoff and Uhlemann, 2006). Consultation with the exhibition aquarium caretaker revealed that the frogs in the present report are a second generation descendant of wild parents, which
M. Haridy et al.
288
originated from forests in Gifu prefecture, and are fed a diet of commercially-produced field crickets. The source of infection was not determined in this study. No further problem in frogs in this aquarium was reported and neither treatment nor destruction of frogs in affected tanks was performed. No other tanks of the same aquarium or other facilities that feed the crickets from the same commercial supplier have reported any similar problems.
Acknowledgement This study was supported in part by a grant-in-aid from Hokkaido University Research Center for Zoonosis Control and JSPS KAKENHI Grant No. 24689034.
References American Thoracic Society (2000) Diagnostic standards and classification of tuberculosis in adults and children. American Journal of Respiratory and Critical Care Medicine, 161, 1376e1395. Asfari M (1988) Mycobacterium-induced infectious granuloma in Xenopus: histopathology and transmissibility. Cancer Research, 48, 958e963. Bouley DM, Ghori N, Mercer KL, Falkow S, Ramakrishnan L (2001) Dynamic nature of hostepathogen interactions in Mycobacterium marinum granulomas. Infection and Immunity, 69, 7820e7831. Brocklebank J, Raverty S, Robinson J (2003) Mycobacteriosis in Atlantic salmon farmed in British Columbia. Canadian Veterinary Journal, 44, 486e489. Broussard GW, Ennis DG (2007) Mycobacterium marinum produces long-term chronic infections in medaka: a new animal model for studying human tuberculosis. Comparative Biochemistry and Physiology, 145, 45e54. Buddle BM, Young LJ (2000) Immunobiology of mycobacterial infections in marsupials. Developmental and Comparative Immunology, 24, 517e529. Chai N, Deforges L, Sougakoff W, Truffot-Pernot C, De Luze A et al. (2006) Mycobacterium szulgai infection in a captive population of African clawed frogs (Xenopus tropicalis). Journal Of Zoo and Wildlife Medicine, 37, 55e58. Cosma CL, Swaim LE, Volkman H, Ramakrishnan L, Davis JM (2006) Zebrafish and frog models of Mycobacterium marinum infection. Current Protocols in Microbiology, 3(10B2), 1e33. Das DK, Pant JN, Chachra KL, Murthy NS, Satyanarayan L et al. (1990) Tuberculous lymphadenitis: correlation of cellular components and necrosis in lymph node aspirate with AFB-positivity and bacillary count. Indian Journal of Pathology and Microbiology, 33, 1e10. Decostere A, Hermans K, Haesebrouck F (2004) Piscine mycobacteriosis: a literature review covering the agent and the disease it causes in fish and humans. Veterinary Microbiology, 99, 159e166.
Devulder G, Perouse de Montclos M, Flandrois JP (2005) A multigene approach to phylogenetic analysis using the genus Mycobacterium as a model. International Journal of Systematic and Evolutionary Microbiology, 55, 293e302. Doig KD, Holt KE, Fyfe JA, Lavender CJ, Eddyani M et al. (2012) On the origin of Mycobacterium ulcerans, the causative agent of Buruli ulcer. BMC Genomics, 13, 258. Ferreira R, Fonseca Lde S, Afonso AM, da Silva MG, Saad MH et al. (2006) A report of mycobacteriosis caused by Mycobacterium marinum in bullfrogs (Rana catesbeiana). Veterinary Journal, 171, 177e180. Fremont-Rahl JJ, Ek C, Williamson HR, Small PL, Fox JG et al. (2011) Mycobacterium liflandii outbreak in a research colony of Xenopus (Silurana) tropicalis frogs. Veterinary Pathology, 48, 856e867. Gauthier DT, Rhodes MW (2009) Mycobacteriosis in fishes: a review. Veterinary Journal, 180, 33e47. Gauthier DT, Rhodes MW, Vogelbein WK, Kator H, Ottinger CA (2003) Experimental mycobacteriosis in striped bass Morone saxatilis. Diseases of Aquatic Organisms, 54, 105e117. Giavenni R, Finazzi M, Poli G, Grimaldi E (1980) Tuberculosis in marine tropical fishes in an aquarium. Journal of Wildlife Diseases, 16, 161e168. Godfrey D, Williamson H, Silverman J, Small PL (2007) Newly identified Mycobacterium species in a Xenopus laevis colony. Comparative Medicine, 57, 97e104. Green SL, Lifland BD, Bouley DM, Brown BA, Wallace RJ Jr. et al. (2000) Disease attributed to Mycobacterium chelonae in South African clawed frogs (Xenopus laevis). Comparative Medicine, 50, 675e679. Iwai N, Kagaya T (2005) Larval food habits of the forest green tree frog. Bulletin of the Herpetological Society of Japan, 2, 100e102. Kaneko Y, Matsui M (2004) Rhacophorus arboreus. In: IUCN 2013. IUCN Red List of Threatened Species. Version 2013.2. www.iucnredlist.org, downloaded April 21st, 2014. Kusano T, Sakai A, Hatanaka S (2006) Ecological functions of the foam nests of the Japanese treefrog, Rhacophorus arboreus (Amphibia, Rhacophoridae). Herpetological Journal, 16, 163e169. Lewis FM, Marsh BJ, von Reyn CF (2003) Fish tank exposure and cutaneous infections due to Mycobacterium marinum: tuberculin skin testing, treatment, and prevention. Clinical Infectious Diseases, 37, 390e397. Muto K, Kubota HY (2011) Ultrastructural analysis of spermiogenesis in Rhacophorus arboreus (Amphibia, Anura, Rhacophoridae). Journal of Morphology, 272, 1422e1434. Nenoff P, Uhlemann R (2006) Mycobacteriosis in mangrove killifish (Rivulus magdalenae) caused by living fish food (Tubifex tubifex) infected with Mycobacterium marinum. Deutsche Tierarztliche Wochenschrift, 113, 230e232. Palys T, Nakamura LK, Cohan FM (1997) Discovery and classification of ecological diversity in the bacterial world: the role of DNA sequence data. International Journal of Systematic Bacteriology, 47, 1145e1156.
Mycobacteriosis in Green Tree Frogs
Ram R, Swarnalatha G, Desai M, Rakesh Y, Uppin M et al. (2011) Membranous nephropathy and granulomatous interstitial nephritis due to tuberculosis. Clinical Nephrology, 76, 487e491. Ramakrishnan L, Valdivia RH, McKerrow JH, Falkow S (1997) Mycobacterium marinum causes both long-term subclinical infection and acute disease in the leopard frog (Rana pipiens). Infection and Immunity, 65, 767e773. Sanchez-Morgado JM, Gallagher A, Johnson LK (2009) Mycobacterium gordonae infection in a colony of African clawed frogs (Xenopus tropicalis). Laboratory Animals, 43, 300e303. Suykerbuyk P, Vleminckx K, Pasmans F, Stragier P, Ablordey A et al. (2007) Mycobacterium liflandii infection in European colony of Silurana tropicalis. Emerging Infectious Diseases, 13, 743e746. Swaim LE, Connolly LE, Volkman HE, Humbert O, Born DE et al. (2006) Mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity. Infection and Immunity, 74, 6108e6117. Talaat AM, Reimschuessel R, Wasserman SS, Trucksis M (1998) Goldfish, Carassius auratus, a novel animal model for the study of Mycobacterium marinum pathogenesis. Infection and Immunity, 66, 2938e2942. Telenti A, Marchesi F, Balz M, Bally F, Bottger EC et al. (1993) Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. Journal of Clinical Microbiology, 31, 175e178. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673e4680.
289
Travis WD, Travis LB, Roberts GD, Su DW, Weiland LW (1985) The histopathologic spectrum in Mycobacterium marinum infection. Archives of Pathology and Laboratory Medicine, 109, 1109e1113. Ulrichs T, Kaufmann SH (2002) Mycobacterial persistence and immunity. Frontiers in Bioscience, 7, d458ed469. Ulrichs T, Kaufmann SH (2006) New insights into the function of granulomas in human tuberculosis. Journal of Pathology, 208, 261e269. Ulrichs T, Kosmiadi GA, Trusov V, Jorg S, Pradl L et al. (2004) Human tuberculous granulomas induce peripheral lymphoid follicle-like structures to orchestrate local host defence in the lung. Journal of Pathology, 204, 217e228. Wilkinson J (2003) Kinugasa flying frog, Rhacophorus arboreus. In: Grzimek’s Animal Life Encyclopedia, Vol. 6, Amphibians, 2nd Edit., M Hutchins, WE Duellman, N Schlager, Eds., Gale Group, Farmington, pp. 291e300. Williams GT, Williams WJ (1983) Granulomatous inflammation e a review. Journal of Clinical Pathology, 36, 723e733. Yamamura Y, Ogawa Y, Maeda H (1974) Prevention of tuberculous cavity formation by desensitization with tuberculin-active peptide. American Review of Respiratory Disease, 109, 594e601. Zhang Y (2004) Persistent and dormant tubercle bacilli and latent tuberculosis. Frontiers in Bioscience, 9, 1136e1156.
January 8th, 2014 ½ Received, Accepted, April 24th, 2014
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
DISEASE IN WILDLIFE OR EXOTIC SPECIES
Mycobacterium marinum Infection in Japanese Forest Green Tree Frogs (Rhacophorus arboreus) M. Haridy*,†, Y. Tachikawa‡, S. Yoshidax,k, K. Tsuyuguchix, M. Tomitax, S. Maeda{, T. Wadak, K. Ibi*, H. Sakai* and T. Yanai* * Department of Pathogenetic Veterinary Sciences, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan, † Department of Pathology and Clinical Pathology, Faculty of Veterinary Medicine, South Valley University, Qena 83523, Egypt, ‡ Gifu World Fresh Water Aquarium, Gifu, x National Hospital Organization, Kinki-Chuo Chest Medical Centre, Sakai, Osaka 591-8555, k Institute of Tropical Medicine, Nagasaki University, Nagasaki and { The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama, Kiyose-shi, Tokyo 204-8533, Japan
Summary Four Japanese forest green tree frogs (Rhacophorus arboreus) were presented with emaciation, abdominal distention and ulcerative and nodular cutaneous lesions affecting the brisket, limbs, digits and ventral abdomen. Another three frogs had been found dead in the same tank 1 year previously. Necropsy examination of these seven frogs revealed splenomegaly and hepatomegaly, with multiple taneyellow nodular foci present in the liver, spleen, heart, lungs, ovaries and kidneys. Microscopically, five frogs had necrosis and surrounding granulomatous inflammation in the liver, spleen, kidneys, lungs, intestine and ovaries, with numerous acid-fast bacilli in the areas of necrosis. Two frogs had granulomatous lesions in the lungs, liver, spleen, heart, coelomic membrane, stomach and intestinal wall. These lesions had no or minimal necrosis and few acid-fast bacilli. Mycobacterium spp. was cultured from three frogs and identified as Mycobacterium marinum by colony growth rate and photochromogenicity and DNA sequencing. This is the first report of M. marinum infection in Japanese forest green tree frogs. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: Japanese forest green tree frog; mycobacteriosis; Mycobacterium marinum; Rhacophorus arboreus
Introduction Japanese forest green tree frogs (Rhacophorus arboreus), designated as a ‘special natural monument’ in Japan, inhabit Honshu Island in forests, near rice fields and in marshy land. They live in mountainous forests as high as 2,000 m. During the breeding season, the frogs can be found near ponds and lakes, but they live primarily in trees in forests and are the only frogs in Japan that lay their eggs in trees. They are nocturnal and native to Japan. Their predators include snakes, weasels, foxes, raccoon dogs and badgers (Wilkinson, 2003). Most studies of Japanese forest green tree frogs have focused on their reproductive biology and ecol-
ogy (Kusano et al., 2006; Muto and Kubota, 2011). Japanese forest green tree frogs have been reported in the International Union for the Conservation of Nature (IUCN) Red List of threatened species as being of ‘least concern’ (Kaneko and Matsui, 2004). Mycobacterium marinum is a slow-growing environmental mycobacterium that is distributed widely in aquatic environments, especially in fish tanks and swimming pools, and is the most common species associated with tuberculous granulomas in aquariumhoused and wild fish (Gauthier and Rhodes, 2009). M. marinum is a zoonotic agent, which may cause granulomatous lesions on the hands and feet of people following exposure of abraded skin to infected water. Deeper spread of infection may lead to tenosynovitis, arthritis, bursitis and osteomyelitis. Systemic infection
*Correspondence to: T. Yanai (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.04.014
Ó 2014 Elsevier Ltd. All rights reserved.
278
M. Haridy et al.
is rare and always occurs in immunosuppressed individuals (Lewis et al., 2003). M. marinum infection is described infrequently in frogs, although frogs, snakes and turtles have been considered a source of infection for fish (Decostere et al., 2004). M. marinum causes chronic granulomatous non-lethal disease in immunocompetent leopard frogs (Rana pipiens) and an acute, fulminant lethal disease in immunosuppressed frogs (Ramakrishnan et al., 1997). M. marinum was also reported as the causative agent of an outbreak of mycobacteriosis in bullfrogs (Rana catesbeiana) in a commercial breeding farm in Brazil. The infection in that outbreak was characterized by skin lesions and disseminated granulomatous lesions in both symptomatic and asymptomatic frogs (Ferreira et al., 2006). Other non-tuberculous mycobacteria, including Mycobacterium chelonae, Mycobacterium xenopi and Mycobacterium liflandii, have been isolated from diseased South African clawed frogs (Xenopus laevis) (Green et al., 2000; Godfrey et al., 2007) and Mycobacterium gordonae, Mycobacterium szulgai and M. liflandii have been isolated from diseased western clawed frogs (Xenopus tropicalis) (Chai et al., 2006; Suykerbuyk et al., 2007; SanchezMorgado et al., 2009; Fremont-Rahl et al., 2011). South African and western clawed frogs are popular laboratory amphibian models. Mycobacterium ulcerans is related genetically to M. marinum and genomic sequencing has confirmed the evolution of M. ulcerans from a common M. marinum progenitor (Doig et al., 2012). The resolution power of 16S rRNA sequences alone is often insufficient for comparing closely-related organisms (Palys et al., 1997). Protein-encoding genes may be more discriminatory than rRNA-encoding genes (Palys et al., 1997). The genes encoding the 65 kD heat shock protein (hsp65) and b subunit of bacterial RNA polymerase (rpoB) are present in all Mycobacterium spp., but there are species-specific variations (Devulder et al., 2005). The production of frog meat by raniculture has become an important economic activity in many countries, yet little is known about frog diseases and the role of frogs in the maintenance and dissemination of zoonotic diseases in production farms and exhibition aquaria. M. marinum infection in frogs and fish is used as a model for the study of human tuberculosis in order to understand the basic aspects of Mycobacteriumehost interaction and granuloma development, as well as trafficking of immune cells in host tissues (Cosma et al., 2006). In the present study, we describe the pathological findings of M. marinum infection in a new host, the Japanese forest green tree frog (R. arboreus), in an exhibition aquarium in Japan.
Materials and Methods Case Material and Clinical History
In September 2013, a dead Japanese forest green tree frog (case 1) was sent from an exhibition aquarium in Gifu Prefecture to the Laboratory of Veterinary Pathology, Gifu University. The frog was emaciated, skinless and had dark, pinpoint, brown discolouration of the musculature. One week later, another three frogs from the same aquarium were presented. One moribund frog had a distended abdomen and died 15 h after arrival (case 2). The other two frogs were in good body condition, but one had ulcerative and nodular cutaneous lesions affecting the brisket, limbs, digits and ventral abdomen (case 3) and the other had no apparent lesions (case 4). Three further frogs had been found dead in the same tank at the exhibition aquarium 1 year previously. These had been preserved in 10% neutral buffered formalin and were submitted at the same time as cases 1e4. One of these fixed frogs had white discolouration of one eye (case 5), but the other two frogs had no external lesions (cases 6 and 7). Histopathology
Tissue samples of the internal organs were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections were stained by haematoxylin and eosin (HE) and by the ZiehleNeelsen (ZN) stain for acid-fast bacteria when mycobacteriosis was suspected. Cytological impression smears of liver, spleen, kidney and coelomic effusion were stained by WrighteGiemsa stain. Bacteriology
Tissue specimens from three frogs (cases 2e4), including liver, kidney and spleen, were removed aseptically for bacteriological examination. Smears of these tissue samples were stained by ZN and the fluorescent auramineerhodamine staining method and the stained specimens were scored according to the recommendations of the American Thoracic Society (2000). The tissue specimens were homogenized in water and treated with N-acetyl-L-cysteinesodium hydroxide for decontamination. They were then inoculated into 1% Ogawa medium (Kyokuto Pharmaceuticals Industrials Co. Ltd, Tokyo, Japan) and a Mycobacterium growth-indicator tube (MGIT) (Becton Dickinson and Co., Franklin Lakes, New Jersey, USA) and incubated at 25 C and 36 C for 8 weeks. The photochromogenicity of the
Mycobacteriosis in Green Tree Frogs
mycobacterial colonies growing on Ogawa medium was checked daily after exposure to light. DNA Sequencing
Several subcultured colonies were suspended in 100 ml of sterile distilled water and heat-denatured in boiling water for 10 min. The suspensions were used as polymerase chain reaction (PCR) templates for sequencing analysis. Four DNA sequences, the entire internal transcribed spacer (ITS), partial 16S rDNA (477 of N terminus), hsp65 (401 base pairs [bp]) and rpoB (397 bp), were determined using methods described elsewhere (Telenti et al., 1993; Devulder et al., 2005). The sequences obtained were compared with sequences from the DNA Data Bank of Japan (DDBJ) using BLAST (http://blast.ddbj.nig.ac.jp/ blastn). The hsp65 and rpoB sequences were also analyzed using CLUSTALW (Thompson et al., 1994) for multiple alignments with those genes from reference strains of M. marinum and M. ulcerans Agy99.
Results Gross Pathology
Case 1 was emaciated with pinpoint dark haemorrhagic foci in the muscles of the back and hindlimbs. Additionally, there was hepatomegaly, mottled discolouration of the liver and kidney and pinpoint white foci in the spleen. Case 2 had a distended abdomen with coelomic effusion, hepatosplenomegaly, a mottled discoloured liver (Fig. 1a) and multiple taneyellow foci in the spleen, heart and ovary (Fig. 1b). Skin lesions in case 3 were multifocal, primarily involving the limbs, dorsum and ventral abdomen. The lesions were discrete, raised, tanered, nodular foci (0.5e1.5 mm) on the dorsum and ventral abdomen (Fig. 1c), as well as smooth tanered glistening abrasions and ulcers on the digits, limbs, brisket and ventral abdomen (Fig. 1d). Multifocal to coalescing tanewhite foci were observed in the peritoneum, liver, spleen and testis (Fig. 1e). Case 4 was apparently healthy; however, necropsy examination revealed the presence of several whiteetan foci in the liver and lungs (Fig. 1f). A few whiteetan pinpoint foci were also observed in the kidney, heart and stomach in case 5. Marked hepatomegaly and prominent yellow foci in the lungs and heart were observed in case 6, and a mottled discoloured liver in case 7. Splenomegaly and hepatomegaly were present in five cases. On cross sectioning, the splenic and hepatic parenchyma had distinct, variably sized, multifocal to coalescing taneyellow and white foci (0.5e2.0 mm).
279
Cytology and Histopathology
Smears of coelomic fluid from two frogs (cases 2 and 5) were moderately cellular. Nucleated cells were predominantly large mononuclear cells interpreted as being macrophages. The macrophages had abundant vacuolated cytoplasm. Numerous rod-shaped to filamentous, negatively-stained, slender, linear bacilli, approximately 5.5 1.25 mm and consistent with Mycobacterium spp., were observed in the cytoplasm of macrophages and extracellularly (Fig. 2a). Multiple foci of necrosis and granulomatous inflammation were observed in the liver, spleen, kidneys, lungs, intestine and ovaries in five cases (1, 2 and 5e7), with abundant acid-fast bacilli in the centre of the lesions (Fig. 2b). These lesions were characterized by a central necrotic core surrounded by few macrophages (Fig. 2c). The periphery of the necrotic core was stained deeply by ZN stain. Dispersed acidfast bacilli were observed in macrophages in the splenic red pulp. The granulomata sometimes appeared as collections of macrophages engulfing numerous bacilli or, alternatively, as small necrotic granulomata constituted mainly of macrophages with a very small necrotic centre containing abundant bacilli (Fig. 2d). The kidney lesions were confined to the interstitium and comprised of either necrotic tissue with minimal macrophage infiltration or a collection of infiltrating cells without necrosis (Fig. 2e). Free bacilli and bacilli within macrophages were observed in the blood vessel lumina. Renal tubules and glomeruli were almost completely free of bacilli. In hepatic tissue, although the predominant lesions were the multiple foci of necrosis and granulomatous inflammation (Fig. 2f), there was also evidence of periductal fibrosis, fatty degeneration and depletion of melanomacrophage centres. A high density of acid-fast bacilli was observed at the junction of the necrotic core with the surrounding cellular zone of macrophages and epithelioid cells (Fig. 2g). Vasculitis, characterized by infiltration of acid-fast bacilli and mononuclear cells into the subintima and tunica media, was also observed (Fig. 2h). Necrosis of blood vessel walls appeared to be a consequence of extension from adjacent tissue inflammatory foci. Emboli of necrotic debris and macrophages containing bacilli were also detected in some blood vessels. Occasional endothelial cells contained acid-fast bacilli. In the lungs, the lesions ranged from focal thickening of the pulmonary interstitium by disorganized collections of macrophages containing acid-fast bacilli to areas of necrosis and granulomatous inflammation replacing the entire interstitium. In cardiac muscles,
M. Haridy et al.
280
a
b
c
d
e
f
Fig. 1. Gross lesions in Japanese forest green tree frogs infected with M. marinum. (a) Hepatomegaly with mottling (case 2). (b) Multiple taneyellow granulomas in the spleen, heart and ovary (case 2). (c and d) Glistening skin ulcers on the dorsum, digits, limbs, brisket and abdomen (case 3). (e) Multiple white foci on the peritoneum, spleen and liver (case 3). (f) Solitary granuloma in the liver and spleen (case 4).
acid-fast bacilli were present within macrophages and were also noted extracellularly in and adjacent to small blood vessels. Tiny granulomas were noted rarely. Haemorrhages between skeletal muscle fibre bundles (case 1) were present, together with macrophages containing acid-fast bacilli in small arteries. In addition to necrotic and granulomatous lesions in the intestinal wall, collections of foamy macrophages were also observed. Acid-fast bacilli were pre-
sent within the necrotic core as well as within the cytoplasm of foamy macrophages. Blood vessels containing macrophage-laden acid-fast bacilli were observed. Degenerate ova with tiny necrotic and granulomatous foci containing macrophages with cytoplasmic acid-fast bacilli were observed in case 2 (Fig. 2i). The lesions observed in the formalin-fixed frogs (cases 5e7) were also characterized by necrosis with
Mycobacteriosis in Green Tree Frogs
a
b
c
d
e
f
g
h
i
j
k
l
281
Fig. 2. Cytological and histopathological appearance of caseous necrotic granulomas in Japanese forest green tree frogs infected with M. marinum. (a) WrighteGiemsa-stained mononuclear cells with abundant vacuolated cytoplasm containing numerous rod-shaped to filamentous, slender and linear bacilli (case 2). Bar, 20 mm. (b) Multiple caseous necrotic granulomata in the spleen showing a central necrotic core (case 2). HE. Bar, 200 mm. (c) Caseous necrotic granuloma in the spleen with minimal surrounding inflammatory cell infiltration (case 2). HE. Bar, 40 mm. (d) Small necrotic granulomas, comprised mainly of macrophages with a small necrotic centre, contain numerous acid-fast bacilli (case 1). ZN. Bar, 40 mm. (e) A lesion of the renal interstitium that is comprised of necrotic tissue with minimal macrophage infiltration (case 1). The area is stained deeply by ZN. Bar, 80 mm. (f) Multiple caseous necrotic granulomata in hepatic tissue (case 1). HE. Bar, 200 mm. (g) A high density of acid-fast bacilli is observed at the periphery of the necrotic core (case 2). ZN. Bar, 40 mm. (h) Vasculitis with infiltration of mononuclear cells lacking acid-fast bacilli in hepatic tissue (case 1). HE. Bar, 40 mm. (i) Degenerate ova with tiny necrotic granulomata in the ovaries (case 2). HE. Bar, 400 mm. (j) A chronic caseous necrotic granuloma encircled by a thick wall of epithelioid cells and fibroblasts (case 5). HE. Bar, 80 mm. (k) Extensive fibroplasia and bile duct proliferation enclosing several melanomacrophage centres associated with multiple extensive areas of necrosis (case 6). HE. Bar, 80 mm. (l) Hypertrophy of the melanomacrophage centres with large foamy macrophages (case 7). HE. Bar, 40 mm.
282
M. Haridy et al.
granulomatous inflammation and extensive fibrosis. In case 5, focal mononuclear cell infiltrations, including macrophages, epithelioid cells and lymphocytes, were observed in the limbus of the eye. Several chronic caseous necrotic granulomas with a thick wall of epithelioid cells and fibrous connective tissue were observed in renal, gastric submucosal and peritoneal tissues (Fig. 2j). Marked destructive caseous necrotic lesions with extensive fibroplasia and bile duct proliferation, enclosing several melanomacrophage centres, were observed in the liver of case 6 (Fig. 2k). Additionally, the lungs and heart of this animal had severe multifocal destructive caseous necrosis. Hypertrophy of the melanomacrophage centres, with large foamy macrophages, was observed in case 7, and sometimes depletion of melanin cells was present (Fig. 2l). Large numbers of acid-fast bacilli were present in the areas of caseous necrosis and in the melanomacrophage centres. Focal lymphocytic infiltration was observed in one or more of the renal, hepatic and cardiac tissues in cases 5, 6 and 7. Mononuclear cell infiltration consisting mainly of lymphocytes was observed in the wall of blood vessels in hepatic tissue. Multifocal epithelioid granulomatous lesions were observed independently in the lungs, liver, kidneys, stomach and intestinal walls, cardiac and skeletal muscles and coelomic membrane in cases 3 and 4. Epithelioid granulomas were characterized by tightly-packed cells with indistinct cytoplasmic borders and abundant eosinophilic cytoplasm with rare or few acid-fast bacilli. There was almost absent or minimal necrosis within these epithelioid granulomas. In the lungs, areas of pulmonary interstitial thickening with epithelioid granulomas appeared as foci of epithelioid cells and occasional foamy macrophages containing few acid-fast bacilli (Fig. 3aec). A few epithelioid granulomas had central necrotic cells with a higher number of acid-fast bacilli. The bacilli were dispersed singly and intracellularly. The pulmonary interstitium was markedly affected and there was focal dystrophic calcification. In the liver, large epithelioid granulomas containing few acid-fast bacilli were associated with enlarged melanomacrophage centres with centrally-located foamy macrophages surrounded by epithelioid cells. These centres contained few or no acid-fast bacilli. There was also extensive epithelioid cell infiltration of the blood vessel wall, some of these cells containing a few acid-fast bacilli. Renal epithelioid granulomas were composed mainly of epithelioid cells and occasional foamy macrophages containing few acid-fast bacilli (Fig. 3d, e). Splenic epithelioid granulomas (Fig. 3f) were composed of mixed proportions of epithelioid cells in the outer layer and foamy macro-
phages containing few acid-fast bacilli in the centre (Fig. 3g). Two large epithelioid granulomas were found in the heart; however, one had a small necrotic centre and relatively more acid-fast bacilli than the other. Granulomatous coelomitis with abundant macrophages and epithelioid cells was observed in case 3 (Fig. 3h). In that animal, the skeletal muscles contained epithelioid granulomas with few acid-fast bacilli (Fig. 3i). In the stomach, a large, solitary granuloma was found in the muscular layer, with a central necrotic core surrounded by foamy macrophages and epithelioid cells containing few acid-fast bacilli. In the tunica muscularis of the intestine, large granulomas composed mainly of macrophages and epithelioid cells with few acid-fast bacilli were observed. Bacteriology and DNA Sequencing
The aetiological agents were cultured from frogs 2, 3 and 4 (Table 1). Several smooth, white colonies (Fig. 4) grew on solid Ogawa medium (incubated at 25 C) within 3 weeks in each case (Table 1). Inoculation into MGIT also led to growth of the causative agents. However, the bacteria in cases 2 and 3 failed to grow at higher temperature (36 C). The colonies turned yellow after exposure to light (photochromogenicity), which is characteristic of M. marinum (Fig. 4). The ITS and 16S rDNA sequences obtained from these organisms were identical to those of a reference strain of M. ulcerans (Agy99) and various strains of M. marinum. The sequences were registered in the GenBank database as AB624295.1 (ITS) and AM884315.1 (16SrDNA) (data not shown). Two housekeeping genes (hsp65 and rpoB) were also analyzed and were shown to be identical in sequence to those of M. marinum, but slightly different to those of M. ulcerans (Fig. 5).
Discussion The present study reports the pathological changes associated with M. marinum infection in Japanese forest green tree frogs. Five frogs had necrotic and granulomatous lesions in the liver, spleen, kidneys, lungs, intestine and ovaries, with numerous acid-fast bacilli in the necrotic centre. In two of these five frogs, a central necrotic core was surrounded by a thin rim of macrophages, while the lesions in the other three animals were more granulomatous in nature and surrounded by a thick wall of epithelioid cells and fibroblastic tissue. Similar epithelioid granulomatous lesions were observed independently in two further frogs in the lungs, liver, spleen, heart, coelomic membrane, stomach and intestinal wall and were
Mycobacteriosis in Green Tree Frogs
a
b
c
d
e
f
g
h
i
283
Fig. 3. Histopathological appearance of epithelioid granulomas in Japanese forest green tree frogs infected with M. marinum. (a) Multifocal epithelioid granulomas in the pulmonary interstitium (case 3). HE. Bar, 400 mm. (b) These are composed mainly of densely-packed epithelioid cells. HE. Bar, 20 mm. (c) They contain few acid-fast bacilli. ZN. Bar, 20 mm. (d) Renal epithelioid granuloma composed primarily of epithelioid cells (case 3). HE. Bar, 40 mm. (e) This contains few acid-fast bacilli. ZN. Bar, 20 mm. (f) Splenic epithelioid granulomata composed of epithelioid and foamy macrophages (case 4). HE. Bar, 200 mm. (g) These contain few acid-fast bacilli. ZN. Bar, 20 mm. (h) Granulomatous coelomitis with abundant macrophages and epithelioid cells (case 3). HE. Bar, 200 mm. (i) Skeletal muscles with an epithelioid granuloma comprised of tightly-packed epithelioid cells (case 3). HE. Bar, 80 mm.
characterized by extensive epithelioid cell formation with no or minimal necrosis and rare or few acidfast bacilli. The discrete well-organized granulomas (‘tuberculoid granulomas’) that occur in human infections with Mycobacterium tuberculosis are usually a result of strong cell-mediated immunity (CMI). Morphologically, tuberculoid granulomas may be simple or complex in organization, usually contain a paucity of bacilli, may or may not have central caseation and are composed predominantly of macrophages, epithelioid macrophages and multinucleated giant cells (Langhans giant cells) that are encircled by lymphocytes, plasma cells and a peripheral rim of fibroblasts
(Williams and Williams, 1983). The epithelioid granulomas caused by M. marinum infection in the present Japanese forest green tree frogs shared characteristics with those of tuberculoid granulomas including a dominant macrophage and epithelioid cell zone containing few acid-fast bacilli, occasionally with peripheral lymphocyte infiltration. No giant cells were observed in any of the frogs examined. Similarly, no giant cells were observed in the granulomas caused by M. marinum and M. liflandii in other species of frogs (Ramakrishnan et al., 1997; Bouley et al., 2001; Ferreira et al., 2006; Fremont-Rahl et al., 2011). The so-called ‘lepromatous granulomas’ arise in a host with robust humoral immunity, but poor CMI
M. Haridy et al.
284 Table 1 Mycobacterial colonies cultured from infected Rhacophorus arboreus 25 C
Case number Specimen Acid-fast stain*
36 C
MGIT Ogawa MGIT Ogawa 2
Spleen Kidney Liver
3+ 3+ 3+
+ + +
+ + +
+ + +
+ +
3
Liver Kidney
2+ 3+
+ +
+ +
4
Liver Kidney
3+ 3+
+ +
+ +
*
ZiehleNeelsen and fluorescent auramineerhodamine staining. For ZN (1,000), a quantitative score of acid-fast bacilli (AFB) was assigned: , no AFB in 300 fields; +, 1e9 AFB in 100 fields; 2+, 1e9 AFB in 10 fields; 3+, 1e9 AFB in one field. For fluorochrome staining (250), quantitative scoring of AFB was used: , no AFB in 30 fields; +, 1e9 AFB in 10 fields; 2+, 1e9 AFB in one field; 3+, 10e90 AFB in one field.
Fig. 4. Morphology (left slant) and photochromogenicity (right slant) of M. marinum colonies on Ogawa medium. White colonies of M. marinum turned yellow when exposed directly to light.
to Mycobacterium spp. These lesions typically lack structural organization and are characterized by diffuse infiltration, mostly of macrophages with numerous intracytoplasmic acid-fast bacilli, sparse lymphocytes and rare to no giant cells (Williams and Williams, 1983). The necrotic granulomata with collections of macrophages containing abundant acid-fast bacilli, and the caseous lesions observed in renal tissue in Japanese forest green tree frogs, shared features of human lepromatous granulomas, including the presence of mature macrophages, epithelioid cells and extracellular components, a fibrous connective tissue capsule and caseation, but the lesions in the frogs lacked giant cells (Bouley et al., 2001). M. marinum can cause caseous necrosis in man, goldfish and the toads Xenopus laevis and Xenopus borealis (Travis et al., 1985; Asfari, 1988; Talaat et al., 1998), but not in leopard frogs (Ramakrishnan et al., 1997; Bouley et al., 2001). In the Japanese forest green tree frogs, the granulomas contained a central caseous necrotic core filled with numerous acid-fast bacilli that was surrounded by a thin rim of epithelioid cells or a thick wall of epithelioid cells and fibroblasts. These granulomas had features similar to those described following experimental inoculation of M. marinum into zebrafish (Swaim et al., 2006), goldfish (Talaat et al., 1998), striped bass (Gauthier et al., 2003) and medaka (Broussard and Ennis, 2007). The epithelioid granulomas were compact and composed of tightly-packed cells that displayed indistinct cytoplasmic borders and abundant eosinophilic cytoplasm, characteristics of epithelioid cells (Bouley et al., 2001). Japanese forest green tree frogs might be a promising animal model for studying tuberculous granulomas because they share both the organized, non-necrotic epithelioid granuloma and the organized necrotic granuloma forms reported in experimental infection with M. marinum of leopard frogs and of zebrafish, respectively. Acid-fast bacilli are observed within the central caseous material and in macrophages of human conventional tuberculous granulomata, and necrotic granulomata containing organisms are seen in active human tuberculosis (Ulrichs and Kaufmann, 2006). Chronic asymptomatic tuberculous infection of man is characterized by fibrotic and calcified granulomas that may or may not contain viable bacteria. Necrosis likely plays an important role in the morbidity and transmission of tuberculosis, but its existence is host and microbial dependant. The necrotic caseous granulomas in Japanese forest green frogs are comparable with the necrotic granulomas of human tuberculosis. A similar high density of acid-fast bacilli is observed in the necrotic centre and epithelioid cell aggregates of
Mycobacteriosis in Green Tree Frogs
285
a
Fig. 5. Multiple alignments of (a) hsp65 and (b) rpoB DNA sequences. Areas of sequence variation are highlighted.
these lesions; however, epithelioid granulomas in frogs also shared some features of human chronic asymptomatic tuberculous infection including low density of acid-fast bacilli, no clinical signs and occasional dystrophic calcification without fibrosis. Measurement of the size of granulomata and calculation of the ratio of the area of the surrounding in-
flammatory cell region to the necrotic core shows that as a granuloma enlarges, the ratio decreases, suggesting that necrosis expands at the expense of the surrounding cell layer rather than being proportional to growth of the granuloma itself (Ulrichs et al., 2004). The necrotic granulomata in the present report had either a wide central necrotic core encircled by a
M. Haridy et al.
286
b
Fig. 5. (continued)
rim of macrophages (cases 1 and 2) or an extensive necrotic area with a thick fibroblastic capsule (case 6), suggesting a late stage of infection. This association was supported by the fatal nature of this type of granuloma in affected frogs. Necrosis within tuberculous granulomas is produced by autodigestion by macrophage enzymes and by a direct toxic action of the causative agent. This process is augmented by
the cellular and humoral immune responses. In the rat model, the main stimulus for necrosis was the formation of immune complexes of antibody and excess bacillus antigen in the centre of lesions. This occurred when CMI was initially strong, but began to decline, allowing the mycobacteria to proliferate out of control of the macrophages. If CMI improved again, the number of bacilli diminished and immune
Mycobacteriosis in Green Tree Frogs
complexes increased, producing an epithelioid granuloma instead of necrosis (Yamamura et al., 1974). The immune status of the frogs in the present report was not examined; however, multifocal areas of lymphocytic infiltration were observed in cases 5, 6 and 7. The acid-fast bacilli in the frogs with necrotic granulomas were found predominantly at the periphery of the necrotic core, adjacent to the epithelioid cell mantle. The same pattern is recognized in human granulomas, where some granulomas harbour mycobacteria within their necrotic cores. Mycobacterium may also be detected in the surrounding region of infiltrating leucocytes, indicating that antigen presentation takes place at the periphery of the classical granuloma structure (Ulrichs et al., 2004). In the present Japanese forest green tree frogs, the presence of epithelioid granulomata characterized by no or minimal necrosis with rare or few acid-fast bacilli suggests a latent infection. A dynamic balance between host immune response and M. tuberculosis is maintained in latent human infections (Ulrichs and Kaufmann, 2002). Dormant M. tuberculosis organisms appear to alter their cell wall structures, since they lose their acid-fast property during latency. In the present study, the tissue sections of the epithelioid and necrotic caseous granulomas of different frogs were stained by ZN at the same time, with the same technique and reagents. This supports a low density of acid-fast bacilli or loss of acid-fast staining of bacteria in epithelioid granulomas. ZN-negative bacilli were detected in chronicallyinfected mice and in non-progressive human tuberculous lung lesions (Zhang, 2004). In the present frogs, lesions in the kidneys were small aggregates of macrophages or tiny necrotic foci confined to the renal interstitium and closely associated with the renal vasculature. Glomerular changes were not detected and both glomeruli and tubules were free of acid-fast bacilli. Small multifocal lymphohistiocytic interstitial aggregates were observed in only one frog out of 23 in a research colony of X. tropicalis infected with M. liflandii (Fremont-Rahl et al., 2011). Tuberculous interstitial nephritis is a common sequela to human renal tuberculosis and glomerular lesions are rarely reported (Ram et al., 2011). In these Japanese forest green tree frogs, acid-fast bacilli were detected in macrophages in the lumina of renal, muscular, cardiac, intestinal and hepatic blood vessels. Extracellular and intracellular acidfast bacilli were observed in the dermal and endomysial vasculature, splenic ellipsoids, renal sinusoids and bronchial vasculature in farmed Atlantic salmon infected with M. chelonae (Brocklebank et al., 2003). Mononuclear cell infiltration of the vascular wall
287
causing vasculitis associated with few or no bacilli was detected in most of the infected frogs. Some endothelial cells of hepatic blood vessels contained acidfast bacilli. Thrombotic lesions associated with numerous mycobacteria have been reported previously in fish with M. marinum infection (Giavenni et al., 1980). Susceptibility to mycobacterial infection depends on the immune status of the host. In immunocompetent leopard frogs inoculated with M. marinum, a chronic granulomatous non-lethal disease was observed. There were at most 1e2 organisms per granuloma. In contrast, leopard frogs administered hydrocortisone intraperitoneally and inoculated with M. marinum exhibited an acute fulminating lethal disease characterized by diffuse chronic inflammatory cell infiltration without granuloma formation or very poorly formed granulomas containing a very high density of M. marinum bacilli (Ramakrishnan et al., 1997). In the present report, necrotic granulomas contained a high density of acid-fast bacilli with little epithelioid recruitment and a typically lethal nature, suggesting severe immunosuppression in the affected frogs. In the other three frogs with an encircling fibrous capsule or fibroplasia, multifocal lymphocytic infiltrations were observed, suggesting that host immunity may be responsible for this encircling of the foci of caseous necrosis with fibroblasts. However, the large number of epithelioid cells in epithelioid granulomas, combined with a scarcity of acid-fast bacilli in these lesions, suggested immunocompetence of the infected frogs. Mycobacterial granulomas in immunosuppressed marsupials may lack such epithelioid and giant cells and fibrous encapsulation (Buddle and Young, 2000). In human tuberculous lymphadenitis, a correlation between the type of granuloma (epithelioid with or without necrosis or necrotic) and the presence and number of acid-fast bacilli revealed that the number of acid-fast bacilli was significantly higher in the presence of necrosis and neutrophilic infiltration when compared with lesions containing lymphocytes, epithelioid cells and Langhans giant cells (Das et al., 1990). Acid-fast bacilli were seen consistently in the intestinal content of frogs 1 and 2, suggesting that food may have been a source of infection. Frogs feed on spiders, insects and sludge worms (Iwai and Kagaya, 2005). M. marinum was demonstrated in the mud tube worm (Tubifex tubifex) that served as live fish food for aquarium fish. The ingestion of these worms by the mangrove killifish was shown to be the source of their infection (Nenoff and Uhlemann, 2006). Consultation with the exhibition aquarium caretaker revealed that the frogs in the present report are a second generation descendant of wild parents, which
M. Haridy et al.
288
originated from forests in Gifu prefecture, and are fed a diet of commercially-produced field crickets. The source of infection was not determined in this study. No further problem in frogs in this aquarium was reported and neither treatment nor destruction of frogs in affected tanks was performed. No other tanks of the same aquarium or other facilities that feed the crickets from the same commercial supplier have reported any similar problems.
Acknowledgement This study was supported in part by a grant-in-aid from Hokkaido University Research Center for Zoonosis Control and JSPS KAKENHI Grant No. 24689034.
References American Thoracic Society (2000) Diagnostic standards and classification of tuberculosis in adults and children. American Journal of Respiratory and Critical Care Medicine, 161, 1376e1395. Asfari M (1988) Mycobacterium-induced infectious granuloma in Xenopus: histopathology and transmissibility. Cancer Research, 48, 958e963. Bouley DM, Ghori N, Mercer KL, Falkow S, Ramakrishnan L (2001) Dynamic nature of hostepathogen interactions in Mycobacterium marinum granulomas. Infection and Immunity, 69, 7820e7831. Brocklebank J, Raverty S, Robinson J (2003) Mycobacteriosis in Atlantic salmon farmed in British Columbia. Canadian Veterinary Journal, 44, 486e489. Broussard GW, Ennis DG (2007) Mycobacterium marinum produces long-term chronic infections in medaka: a new animal model for studying human tuberculosis. Comparative Biochemistry and Physiology, 145, 45e54. Buddle BM, Young LJ (2000) Immunobiology of mycobacterial infections in marsupials. Developmental and Comparative Immunology, 24, 517e529. Chai N, Deforges L, Sougakoff W, Truffot-Pernot C, De Luze A et al. (2006) Mycobacterium szulgai infection in a captive population of African clawed frogs (Xenopus tropicalis). Journal Of Zoo and Wildlife Medicine, 37, 55e58. Cosma CL, Swaim LE, Volkman H, Ramakrishnan L, Davis JM (2006) Zebrafish and frog models of Mycobacterium marinum infection. Current Protocols in Microbiology, 3(10B2), 1e33. Das DK, Pant JN, Chachra KL, Murthy NS, Satyanarayan L et al. (1990) Tuberculous lymphadenitis: correlation of cellular components and necrosis in lymph node aspirate with AFB-positivity and bacillary count. Indian Journal of Pathology and Microbiology, 33, 1e10. Decostere A, Hermans K, Haesebrouck F (2004) Piscine mycobacteriosis: a literature review covering the agent and the disease it causes in fish and humans. Veterinary Microbiology, 99, 159e166.
Devulder G, Perouse de Montclos M, Flandrois JP (2005) A multigene approach to phylogenetic analysis using the genus Mycobacterium as a model. International Journal of Systematic and Evolutionary Microbiology, 55, 293e302. Doig KD, Holt KE, Fyfe JA, Lavender CJ, Eddyani M et al. (2012) On the origin of Mycobacterium ulcerans, the causative agent of Buruli ulcer. BMC Genomics, 13, 258. Ferreira R, Fonseca Lde S, Afonso AM, da Silva MG, Saad MH et al. (2006) A report of mycobacteriosis caused by Mycobacterium marinum in bullfrogs (Rana catesbeiana). Veterinary Journal, 171, 177e180. Fremont-Rahl JJ, Ek C, Williamson HR, Small PL, Fox JG et al. (2011) Mycobacterium liflandii outbreak in a research colony of Xenopus (Silurana) tropicalis frogs. Veterinary Pathology, 48, 856e867. Gauthier DT, Rhodes MW (2009) Mycobacteriosis in fishes: a review. Veterinary Journal, 180, 33e47. Gauthier DT, Rhodes MW, Vogelbein WK, Kator H, Ottinger CA (2003) Experimental mycobacteriosis in striped bass Morone saxatilis. Diseases of Aquatic Organisms, 54, 105e117. Giavenni R, Finazzi M, Poli G, Grimaldi E (1980) Tuberculosis in marine tropical fishes in an aquarium. Journal of Wildlife Diseases, 16, 161e168. Godfrey D, Williamson H, Silverman J, Small PL (2007) Newly identified Mycobacterium species in a Xenopus laevis colony. Comparative Medicine, 57, 97e104. Green SL, Lifland BD, Bouley DM, Brown BA, Wallace RJ Jr. et al. (2000) Disease attributed to Mycobacterium chelonae in South African clawed frogs (Xenopus laevis). Comparative Medicine, 50, 675e679. Iwai N, Kagaya T (2005) Larval food habits of the forest green tree frog. Bulletin of the Herpetological Society of Japan, 2, 100e102. Kaneko Y, Matsui M (2004) Rhacophorus arboreus. In: IUCN 2013. IUCN Red List of Threatened Species. Version 2013.2. www.iucnredlist.org, downloaded April 21st, 2014. Kusano T, Sakai A, Hatanaka S (2006) Ecological functions of the foam nests of the Japanese treefrog, Rhacophorus arboreus (Amphibia, Rhacophoridae). Herpetological Journal, 16, 163e169. Lewis FM, Marsh BJ, von Reyn CF (2003) Fish tank exposure and cutaneous infections due to Mycobacterium marinum: tuberculin skin testing, treatment, and prevention. Clinical Infectious Diseases, 37, 390e397. Muto K, Kubota HY (2011) Ultrastructural analysis of spermiogenesis in Rhacophorus arboreus (Amphibia, Anura, Rhacophoridae). Journal of Morphology, 272, 1422e1434. Nenoff P, Uhlemann R (2006) Mycobacteriosis in mangrove killifish (Rivulus magdalenae) caused by living fish food (Tubifex tubifex) infected with Mycobacterium marinum. Deutsche Tierarztliche Wochenschrift, 113, 230e232. Palys T, Nakamura LK, Cohan FM (1997) Discovery and classification of ecological diversity in the bacterial world: the role of DNA sequence data. International Journal of Systematic Bacteriology, 47, 1145e1156.
Mycobacteriosis in Green Tree Frogs
Ram R, Swarnalatha G, Desai M, Rakesh Y, Uppin M et al. (2011) Membranous nephropathy and granulomatous interstitial nephritis due to tuberculosis. Clinical Nephrology, 76, 487e491. Ramakrishnan L, Valdivia RH, McKerrow JH, Falkow S (1997) Mycobacterium marinum causes both long-term subclinical infection and acute disease in the leopard frog (Rana pipiens). Infection and Immunity, 65, 767e773. Sanchez-Morgado JM, Gallagher A, Johnson LK (2009) Mycobacterium gordonae infection in a colony of African clawed frogs (Xenopus tropicalis). Laboratory Animals, 43, 300e303. Suykerbuyk P, Vleminckx K, Pasmans F, Stragier P, Ablordey A et al. (2007) Mycobacterium liflandii infection in European colony of Silurana tropicalis. Emerging Infectious Diseases, 13, 743e746. Swaim LE, Connolly LE, Volkman HE, Humbert O, Born DE et al. (2006) Mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity. Infection and Immunity, 74, 6108e6117. Talaat AM, Reimschuessel R, Wasserman SS, Trucksis M (1998) Goldfish, Carassius auratus, a novel animal model for the study of Mycobacterium marinum pathogenesis. Infection and Immunity, 66, 2938e2942. Telenti A, Marchesi F, Balz M, Bally F, Bottger EC et al. (1993) Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. Journal of Clinical Microbiology, 31, 175e178. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673e4680.
289
Travis WD, Travis LB, Roberts GD, Su DW, Weiland LW (1985) The histopathologic spectrum in Mycobacterium marinum infection. Archives of Pathology and Laboratory Medicine, 109, 1109e1113. Ulrichs T, Kaufmann SH (2002) Mycobacterial persistence and immunity. Frontiers in Bioscience, 7, d458ed469. Ulrichs T, Kaufmann SH (2006) New insights into the function of granulomas in human tuberculosis. Journal of Pathology, 208, 261e269. Ulrichs T, Kosmiadi GA, Trusov V, Jorg S, Pradl L et al. (2004) Human tuberculous granulomas induce peripheral lymphoid follicle-like structures to orchestrate local host defence in the lung. Journal of Pathology, 204, 217e228. Wilkinson J (2003) Kinugasa flying frog, Rhacophorus arboreus. In: Grzimek’s Animal Life Encyclopedia, Vol. 6, Amphibians, 2nd Edit., M Hutchins, WE Duellman, N Schlager, Eds., Gale Group, Farmington, pp. 291e300. Williams GT, Williams WJ (1983) Granulomatous inflammation e a review. Journal of Clinical Pathology, 36, 723e733. Yamamura Y, Ogawa Y, Maeda H (1974) Prevention of tuberculous cavity formation by desensitization with tuberculin-active peptide. American Review of Respiratory Disease, 109, 594e601. Zhang Y (2004) Persistent and dormant tubercle bacilli and latent tuberculosis. Frontiers in Bioscience, 9, 1136e1156.
January 8th, 2014 ½ Received, Accepted, April 24th, 2014